Approximately 1% of children are born with a congenital heart defect, with half requiring medical and/or surgical treatment. Although survival for these children has improved they continue to suffer morbidity and late mortality. This is due to the fact that they are at great risk for developing pulmonary vascular disease. In fact, even early pulmonary endothelial dysfunction, with abnormal vascular reactivity, causes significant morbidity and mortality. Our recent studies, using or Shunt model of congenital heart disease and increased pulmonary blood flow (PBF), indicate that the one of the major components of the endothelial dysfunction is a reduction in nitric oxide (NO) signaling due to the uncoupling of endothelial NOS. However, the mechanisms by which eNOS becomes uncoupled are incompletely understood. We have shown that eNOS becomes uncoupled, at least in part, due to a reduction in the NOS substrate, L-arginine secondary to an increase in arginase activity. In the prior funding period we demonstrated that acute increases in shear stress utilized H2O2 to stimulate NO signaling. However, although the sustained increase in shear stress in Shunt lambs also leads to increases in H2O2, this correlates with NOS uncoupling, decreased bioavailable NO and increased NO scavenging as peroxynitrite, indicating that H2O2 can also attenuate NO signaling. In this competitive renewal we will elucidate the mechanisms by which sustained increases in shear stress utilizes H2O2 to reduce NO signaling. Based on pilot data, the central hypothesis we will test is that a key event in the disruption of NO signaling in children born with congenital heart disease and increased PBF is a shear stress-mediated decrease in L- arginine. Further, we will determine if this is mediated through an H2O2 dependent increase in RhoA-Rho kinase (ROCK)-signaling that leads to the post-translational activation of arginase. We anticipate that the successful completion of our studies will significantly increase our understanding of the mechanisms underlying the diminished NO-signaling and endothelial dysfunction associated with congenital heart defects that result increased PBF. Further, our studies should identify new signaling pathways that may be amenable to therapeutic intervention.